Telomerase Dysfunction in the Tumorigenesis of Genetic Disorders

Telomeres are nucleoprotein complexes present at the ends of chromosome to maintain its integrity. Telomere length is maintained by an enzyme called "telomerase". Thus, telomerase activity and telomere length are crucial for the initiation of cancer and tumors survival. Also, oxidative stress will cause DNA, protein, and/or lipid damage, which end with changes in chromosome instability, genetic mutation, and may affect cell growth and lead to cancer. Some genetic diseases such as chromosomal instability syndrome, overgrowth syndrome, and neurofibromatosis make the patients at higher risk for developing different types of cancers. Therefore, we aimed to estimate telomerase activity and oxidative stress in these patients. Blood samples were collected from 31 patients (10 with neurofibromatosis, 11 with chromosomal breakage, and 10 with overgrowth syndrome) and 12 healthy subjects. Blood hTERT mRNA was detected by real time quantitative reverse-transcription PCR (RT-qPCR). All patients were subjected to chromosomal examination and chromosome breakage study using diepoxybutane method. Moreover, serum glutathione (GSH), glutathione-s-transferase (GST) activity and nitric oxide (NO) levels were measured among the control and patients groups. Receiver operating characteristic (ROC) curve was drawn to evaluate the efficiency of telomerase activity as a biomarker for the prediction of cancer occurrence. The relative telomerase activity in neurofibromatosis patients was significantly higher than controls (P = 0.014), while it was non-significantly higher in chromosomal breakage and overgrowth patients (P = 0.424 and 0.129, respectively). NO levels in neurofibromatosis, chromosomal breakage and overgrowth patients significantly increased with respect to control (P = 0.021, 0.002, 0.050, respectively). GSH levels were non-significantly lower in neurofibromatosis and chromosomal breakage patients in comparison with the control group, while it remained unchanged in overgrowth patients. The GST activity was significantly upregulated in neurofibromatosis, chromosomal breakage and overgrowth groups in comparison with the control group (P = 0.001, 0.009, and 0.025, respectively). Chromosomal examination revealed normal karyotype in all four chromosomal breakage patients with positive diepoxybutane test. The results of the present study revealed altered telomerase activity and oxidative stress in the studied genetic disorders. More research studies with a larger number of patients are required to confirm whether this alteration is related to cancer occurrence risk or not.

to estimate telomerase activity and oxidative stress in these patients. Blood samples were collected from 31 patients (10 with neurofibromatosis, 11 with chromosomal breakage, and 10 with overgrowth syndrome) and 12 healthy subjects. Blood hTERT mRNA was detected by real time quantitative reverse-transcription PCR (RT-qPCR). All patients were subjected to chromosomal examination and chromosome breakage study using diepoxybutane method. Moreover, serum glutathione (GSH), glutathione-s-transferase (GST) activity and nitric oxide (NO) levels were measured among the control and patients groups. Receiver operating characteristic (ROC) curve was drawn to evaluate the efficiency of telomerase activity as a biomarker for the prediction of cancer occurrence. The relative telomerase activity in neurofibromatosis patients was significantly higher than controls (P = 0.014), while it was non-significantly higher in chromosomal breakage and overgrowth patients (P = 0.424 and 0.129, respectively). NO levels in neurofibromatosis, chromosomal breakage and overgrowth patients significantly increased with respect to control (P = 0.021, 0.002, 0.050, respectively). GSH levels were non-significantly lower in neurofibromatosis and chromosomal breakage patients in comparison with the control group, while it remained unchanged in overgrowth patients. The GST activity was significantly upregulated in neurofibromatosis, chromosomal breakage and overgrowth groups in comparison with the control group (P = 0.001, 0.009, and 0.025, respectively). Chromosomal examination revealed normal karyotype in all four chromosomal breakage patients with positive diepoxybutane test. The results of the present study revealed altered telomerase activity and oxidative stress in the studied genetic disorders. More research studies with a larger number of patients are required to confirm whether this alteration is related to cancer occurrence risk or not.  (20).
The aim of the current study was to assess the telomerase activity as one of the factors that affect telomere function in some groups of tumorigenic genetic disorders compared to the control, and also evaluate the oxidative stress biomarkers as a factor that can affect telomere function in these genetic disorder groups in comparison to healthy controls.

Sample collection
Ten mL of venous blood was collected and distributed into tubes as the following: two ml was delivered under aseptic conditions into a plastic tube containing ethylene diamine tetra acetic acid (EDTA) at concentration of (1.

Chromosomal examination
Chromosomal examination was performed from peripheral blood lymphocytes for all patients to detect chromosomal breakage (25).

Real time PCR quantification
To determine hTERT mRNA levels, qRT-PCR assays were constructed using TaqMan Gene  (28).

Colorimetric determination of nitric oxide level
In acid medium and in the presence of nitrite the formed nitrous acid and the end product are coupled with ethylenediamine. The resulting azo dye has a bright reddish-purple color which can be measured at 540 nm (29). NO level was measured in duplicate using NO assay kit (Biodiagnostic 2000, Egypt).

Determination of the plasma glutathione
The method is based on the reduction of (2nitrobenzoic acid) (DTNB) with GSH to produce a yellow compound. The reduced chromogen is directly proportional to GSH concentration, and its absorbance can be measured at 405 nm (30). GSH of all study subjects was determined in duplicate by GSH reduced kit (Biodiagnostic 2000, Egypt).

Estimation of glutathione S-transferase activity
Total GST activity was estimated by measuring the conjugation of 1-chloro-2,4dinitrobenene (CDNB) with reduced GSH. The conjugation is accompanied by an increase in absorbance at 340 nm. The increase rate is directly proportional to the GST activity in the sample (31).
GST activity was measured in duplicate using GST kit (Biodagnostic 2000, Egypt).

Statistical analysis
Data were statistically analyzed using SPSS

Demographic data
Control group included 12 healthy age and sex     comparison with the control group ( Figure 4).

Gene expression evaluation
Telomerase activity assessment is represented in figure 5 significantly higher telomerase activity  Telomerase activity in overgrowth patients showed non-significant positive correlation with age, GSH and GST levels (R = 0.241, 0.050, and 0.017 respectively), and a non-significant inverse correlation with total leukocyte count and NO levels (R = 0.160, and 0.033, respectively) ( Table 3).

ROC curve analysis
ROC curve analysis showed that telomerase activity has an area under the curve (AUC) value of    Not only the genetic factors, but also many environmental factors may affect telomeres length.
Telomeres are highly susceptible to damage due to oxidative stress (36). Different studies have shown that oxidative stress may partially contribute to telomerase activation. An imbalance between oxidants and antioxidants in favor of oxidants is associated with increased levels of telomerase (37).
Oxidative stress may exert a role in several genetic disorders characterized by neurological degeneration cancer and high cancer prevalence, but may also indicate new strategies for treatment of these syndromes (38).
The present study found that NO levels in neurofibromatosis, chromosomal breakage, and overgrowth patients were significantly higher than the controls. In the same context, many studies reported elevation of the serum NO levels in different types of cancer (39). It is well known that the cells of ataxia telangiectasia patients are particularly sensitive to ROS-generating agents (e.g., hydrogen peroxide and NO) resulting in oxidative stress and oxidative damage to important biomacromolecules and cell structures (40).